The overall objective of this research is to understand the regulated biosynthesis of parathyroid hormone (PTH). The activity of the parathyroid gland is controlled by extracellular concentrations of calcium. In the proposed research we will obtain information about the structure of PTH mRNA and the PTH gene, analyze the effects of calcium on the metabolism of PTH mRNA in parathyroid cells and develop a cell-free system to study the biosynthesis of PTH mRNA. PTH mRNA can be purified to greater than 50% purity and this mRNA will be used as a template to produce DNA complementary to PTH mRNA (cDNA). The cDNA will be enzymatically converted to double-stranded DNA with DNA polymerase and PTH cDNA will be isolated and amplified by molecular cloning. The structure of cDNA will be analyzed by restriction enzyme mapping and sequence analysis. Fragments of genomic DNA containing the PTH gene will then be recovered by molecular cloning and the structure of the genomic gene will be compared with that of cDNA. The cloned cDNA will be used asa probe to analyzed the biosynthesis and contcentrations of PTH mRNA in parathyroid cells exposed to high and low calcium concentrations. The cloned genomic DNA will be used to reconstitute PTH chromatin so that biosynthesis of PTH mRNA can be studied in vitro. The initial translational product of PTH mRNA is a precursor of PTH called pre-ProPTH. We plan to study the metabolism of the precursor by solubilizing and isolating from microsomal membranes the enzymes necessary for the removal and degradation of the presequence. The formation of membrane-bound polysomes may be assayed in vitro by the binding of PTH mRNA to microsomal membranes. We plan to exploit this assay to examine the role of the presequence in initiating the binding of the polysomes to the membrane.